This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific Aims This COBRE project originally had two aims focused on E. histolytica phagocytosis, which are now funded by an NIAID R01 grant. Accordingly, the project's focus and specific aims were changed at the beginning of last year. Leishmanolysin is the founding member of the M8 family of metallo-endopeptidases. It is an immunodominant surface protein of leishmania promastigotes that degrades complement and immunoglobulins. Trypanosoma brucei homologues cleave variant surface glycoproteins during antigenic switching. Interestingly, the Drosophila orthologue invadolysin has been shown to play critical roles in cell division and cell migration. We identified two leishmanolysin homologues in the E. histolytica genome, which we named E. histolytica metallosurface proteases 1 and 2 (EhMSP-1 and -2). The EhMSP-1 gene is not present in Entamoeba dispar, a non-pathogenic ameba that is closely related to E. histolytica. Few differences in the protein coding regions have been identified between E. histolytica and E. dispar. This and the importance of the homologous proteins in leishmania and trypanosome virulence make it important to determine the function of EhMSP-1 and the specific role(s) it plays in E. histolytica biology. Aim 1: Determine if EhMSP-1 is a cell surface metalloproteinase, and, if so, its substrate specificity. Aim 2: Test the hypothesis that EhMSP-1 contributes to immune resistance by degrading IgG, converting the complement protein C3b to iC3b, and cleaving cell surface amebic antigens. Aim 3: Test the hypotheses that EhMSP-1 and EhMSP-2 function in cell division and migration. B. Studies and Results Aim 1: We have purified full-length EhMSP-1 with a 6xhistidine tag expressed in Escherichia coli. This recombinant protein did not have enzymatic activity when assessed by zymography, but has enabled us to: 1) assess immunogenicity of EhMSP-1 in humans naturally infected with E. histolytica using existing serum samples and an ELISA;2) contract a commercial vendor to make rabbit polyclonal anti-EhMSP-1 antibodies, which we have affinity purified;and 3) select several recombinant anti-EhMSP-1 single-chain Fv (scFV) reagents by phage display. EhMSP-1 is immunogenic for humans infected with E. histolytica, since pre-existing serum from nine of twelve individuals with anti-E. histolytica IgG detectable using a commercial kit were positive in our EhMSP-1 ELISA. All negative controls were also negative for anti-EhMSP-1 IgG. To determine if EhMSP-1 is truly a metalloproteinase, we used one of our scFv reagents to immunoprecipitate the protein from wild-type E. histolytica and a commercially available protease assay (the Pierce QuantiCleave assay). Specific immunoprecipitation of EhMSP-1 was confirmed by Western blot. Protease activity was readily detected and was reduced to background levels by the Zn2+ chelator phenanthroline. These data indicate that EhMSP-1 is truly a metalloproteinase. Of note, two bands are seen on immunoblots of E. histolytica lysates. M8-type metalloendopeptidases most often exist in a proform, which are activated by cleavage. This raises the possibility that the lower band is a processed, active form. We are currently trying to determine if this is the case (see below), and are continuing to try to make an active recombinant enzyme. Aim 2: Expression of EhMSP-1 appears to be tightly regulated, since attempts to over-express the protein using a constitutive expression system and attempts to interfere with its expression using a constitutive RNAi method have achieved only short-term perturbations in EhMSP-1 expression. It takes greater than one month to recover transfectants and we only get about two weeks to work with them, so this has severely limited our ability to conduct phenotypic assays. We have transiently reduced EhMSP-1 expression by RNAi. Preliminary experiments using these parasites and a flow cytometry-based assay for inactivation of complement C3b by conversion to iC3b (i.e., staining with an anti-iC3b mouse antibody after incubation with C8-depleted human serum and a complement fixing rabbit antibody) showed that E. histolytica degrades C3b to iC3b, and that trophozoites with reduced EhMSP-1 expression degrade C3b less efficiently. We are working to achieve robust and sustained EhMSP-1 silencing using a recently developed epigenetic method. Since the gene may be essential, we are also developing a tetracycline-inducible vector for regulated EhMSP-1 expression to introduce prior to interfering with expression of the endogenous gene. Aim 3: Using immunofluorescent confocal microscopy, we found that EhMSP-1 is localized to vesicles in all amebae, but is present on the surface of only a subset. Presence of high and low EhMSP-1 surface expressers was confirmed by flow cytometry. We selected multiple clones by growth in agar and analyzed cell surface EhMSP-1 exposure in the recovered populations. In each case, a mixed population of high and low surface expressers was obtained, indicating that the phenomenon is not clonal. This apparent regulation of EhMSP-1 surface exposure led us to hypothesize that the EhMSPs function in cell division like their Drosophila orthologue invadolysin. We have now used serum starvation to synchronize E. histolytica cultures, and shown: 1) that the entire synchronized population has similar EhMSP-1 surface expression (as opposed to the distinct high- and low-expressing populations previously seen);and 2) that surface EhMSP-1 exposure varies systematically during the cell cycle, with lowest levels just prior to cell division. C. Significance: Since EhMSP-1 is one of only a few E. histolytica protein coding sequences absent from E. dispar, understanding its function may provide key insights into E. histolytica virulence. These studies have confirmed that EhMSP-1 is a cell surface metalloproteinase, and they will determine if it is important for immune evasion. Unlike leishmania and the trypansomes, E. dispar is a non-pathogen and E. histolytica is usually non-invasive;this strongly suggests that the EhMSP proteins also have non-virulence related functions. Aim 3 is designed to elucidate these functions, and may suggest unknown functions of leishmanolysin. These studies also may lead to improved methods to prevent and diagnose E. histolytica infection. Since EhMSP-1 is immunogenic in humans, it may be an excellent target for vaccine development. D. Plans: The aims will remain unchanged during the coming year and a manuscript reporting our initial findings is nearly ready for submission. For aim 1, we are focused on making an enzymatically active recombinant protease (by using refolding methods or expressing the extracellular domain, and, if necessary, by expression in Pichia pastoris), which is necessary to determine the substrate specificity of EhMSP-1. We hope to combine this protein with a protease assay to enable selection of scFv antibodies that inhibit protease activity. These antibodies would be used for epitope mapping, and as a tool to help determine the role(s) EhMSP-1 plays in virulence associated phenotypes. For aim 2, we are focused on achieving stable EhMSP-1 silencing or tetracycline regulated expression. This will enable us to rigorously test whether EhMSP-1 participates in degradation of C3b and/or resistance to complement mediated lysis. Our focus in aim 3 is to conduct growth assays using EhMSP-1 knockdown cells, and to use trophozoites expressing GFP-EhMSP-1 fusions for time-lapse microscopy to study EhMSP-1's distribution during cell division and migration. E. Publications (since the 2009 progress report) 1.David R. Tribble, Shahida Baqar, Marya Carmolli, Chad Porter, Catherine J. Larsson, Patricia Guerry, Kristen Pierce, Katrin Sadigh, David Rockabrand, Frederic Poly, Cassandra Ventone, Patrick Daunais, Sandra Dakdouk, Caroline Lyon, Ann Fingar, Christopher Huston, Theron Gilliland Jr., Ericka Jones, Michael Darsley, Beth D. Kirkpatrick. Campylobacter jejuni strain CG8421: A refined model for the study of campylobacteriosis and evaluation of Campylobacter vaccines in human subjects. Clinical Infectious Diseases. 2009. 49:1512-1519. Vertebrate Animals (Item B) No experiments using vertebrate animals are planned. Change is checked on form page 5 because animal experiments were included in the original application, which is now funded by an NIAID R01 grant. As noted, the focus and aims of this project have been changed to avoid scientific overlap. Animal experiments are not necessary for the current aims.